Compounds, compositions and methods for treating or preventing hypoxic or ischemic injury

ABSTRACT

Compositions and methods of treating or preventing disease or injury to a human patient or biological material undergoing ischemic or hypoxic conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. PatentApplication Ser. No. 61/303,532, filed Feb. 11, 2010, which is herebyincorporated by reference in its entirety.

BACKGROUND

Treatment with hydrogen sulfide (H₂S) protects biological matter fromhypoxic and ischemic injury using a stable composition of hydrogensulfide in mammals. (US Pub. No. 2008/0199541). This discovery providesexciting possibilities for the treatment or prevention of a number ofanimal and human diseases, particularly hypoxia and ischemia-relateddiseases and injuries using sulfide compounds.

The present invention meets this need by providing pharmaceuticalcompositions which are demonstrated herein to protect animals frominjury and death resulting from hypoxic and/or ischemic conditions, aswell as other injuries and disease conditions.

SUMMARY

An aspect of the invention is a compound according to the structure (I):

or a specific stereoisomer thereof according to the structure:

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein m is 3or 4; Z is selected from H, CH₃, and CH₂CH₃; or, a salt, ester, hydrate,solvate, or stereoisomer thereof, with the proviso that when m is 3, Zis H, X and Y are C═O, R³ and R⁴ are CH₂, and R⁵ and R⁶ are OH, then R¹and R² are not C₁₋₁₀ alkyl. The polysulfide compound may be symmetricalor asymmetrical. The compound is an active pharmaceutical ingredient(API) useful in various forms of treatment as set forth herein.

Another aspect of the invention is a pharmaceutical compositioncomprising a therapeutically effective dose of a compound according tothe structure (I)

or a specific stereoisomer thereof according to the structure:

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein m is 3or 4; Z is selected from H, CH₃, and CH₂CH₃; or, a salt, ester, hydrate,solvate, or stereoisomer thereof, and, a carrier system comprising oneor more inactive pharmaceutical ingredients, wherein the pharmaceuticalcomposition has a pH of 4.0-7.0 or 5.0-6.5.

Another aspect of the invention is a method for treating or preventinginjury to a biological material exposed to hypoxic or ischemicconditions comprising parenterally administering to the biologicalmaterial or a patient in need thereof a therapeutically effective doseof a compound according to the structure (I):

or a specific stereoisomer thereof according to the structure:

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein m is 3or 4; Z is selected from H, CH₃, and CH₂CH₃; or, a salt, ester, hydrate,solvate or stereoisomer thereof.

Another aspect of the invention is a method of making an intravenous orsubcutaneous dosage form comprising a) providing a compound according tothe structure (I):

or a specific stereoisomer thereof according to the structure:

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein m is 3or 4; Z is selected from H, CH₃, and CH₂CH₃; or, a salt, ester, hydrate,solvate or stereoisomer thereof; b) making a vehicle solution by mixinga solution of 1% citric acid in sterile water with saline, and adding0.5N NaOH in saline to adjust the pH to 5.5-6.5; c) dissolving thepre-determined amount of the compound in the vehicle solution; d)readjusting the pH of this compound-containing solution back to 5.0-6.5or 4.0-7.0 by adding NaOH while stirring; e) and adding a defined secondamount of the vehicle solution to produce an intravenous or subcutaneousdosage form having a predetermined dose concentration.

Another aspect of the invention is a compound according to the structure(II):

or a specific stereoisomer thereof according to the structure:

wherein R¹⁰, R¹¹ and R¹² are each independently a side chain of anaturally-occurring or a non-naturally occurring amino acid (e.g.,CH₂CO₂H, (CH₂)₂CO₂H), a branched or straight chain C₁₋₁₀ alkyl, astraight or branched chain C₁₋₁₀ alkyl substituted with —ONO₂, or, astraight or branched chain C₁₋₁₀ alkyl substituted with —OH; wherein R¹³and R¹⁴ are independently a branched or straight chain C₁₋₁₀ alkanediyl;and, wherein X is independently C═O or SO₂; or, a salt, ester, hydrate,solvate or stereoisomer thereof.

Another aspect of the invention is a pharmaceutical compositioncomprising a therapeutically effective dose of a compound according tothe structure (II):

or a specific stereoisomer thereof according to the structure:

wherein R¹⁰, R¹¹ and R¹² are independently a side chain of anaturally-occurring or a non-naturally occurring amino acid (e.g.,CH₂CO₂H, (CH₂)₂CO₂H), a branched or straight chain C₁₋₁₀ alkyl, astraight or branched chain C₁₋₁₀ alkyl substituted with —ONO₂, or, astraight or branched chain C₁₋₁₀ alkyl substituted with —OH; wherein R¹³and R¹⁴ are independently a branched or straight chain C₁₋₁₀ alkanediyl;and, wherein X is independently C═O or SO₂; or, a salt, ester, hydrate,solvate or stereoisomer thereof; and, a parenteral carrier systemcomprising one or more inactive pharmaceutical ingredients, wherein thepharmaceutical composition has a pH of 4.0-7.0 or 5.0-6.5.

Another aspect of the invention is a method for treating or preventinginjury to a biological material exposed to hypoxic or ischemicconditions comprising parenterally administering to the biologicalmaterial or a patient in need thereof a therapeutically effective doseof a compound according to the structure (II):

or a specific stereoisomer thereof according to the structure:

wherein R¹⁰, R¹¹ and R¹² are independently a side chain of anaturally-occurring or a non-naturally occurring amino acid (e.g.,CH₂CO₂H, (CH₂)₂CO₂H), a branched or straight chain C₁₋₁₀ alkyl, astraight or branched chain C₁₋₁₀ alkyl substituted with —ONO₂, or, astraight or branched chain C₁₋₁₀ alkyl substituted with —OH; wherein R¹³and R¹⁴ are independently a branched or straight chain C₁₋₁₀ alkanediyl;and, wherein X is independently C═O or SO₂; or, a salt, ester, hydrate,solvate or stereoisomer thereof.

Another aspect of the invention is a compound according to the structure(III):

or a specific stereoisomer thereof according to the structure:

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are each independently a side chain of anaturally-occurring or a non-naturally occurring amino acid (e.g.,CH₂CO₂H, (CH₂)₂CO₂H), a branched or straight chain C₁₋₁₀ alkyl, astraight or branched chain C₁₋₁₀ alkyl substituted with —ONO₂, or, astraight or branched chain C₁₋₁₀ alkyl substituted with —OH; wherein R¹⁹and R²⁰ are each independently a branched or straight chain C₁₋₁₀alkanediyl; and, wherein each X is independently C═O or SO₂; or, a salt,ester, hydrate, solvate, or stereoisomer thereof.

Another aspect of the invention is a pharmaceutical compositioncomprising a therapeutically effective dose of a compound according tothe structure (III):

or a specific stereoisomer thereof according to the structure:

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are independently a side chain of anaturally-occurring or a non-naturally occurring amino acid (e.g.,CH₂CO₂H, (CH₂)₂CO₂H), a branched or straight chain C₁₋₁₀ alkyl, astraight or branched chain C₁₋₁₀ alkyl substituted with —ONO₂, or, astraight or branched chain C₁₋₁₀ alkyl substituted with —OH; wherein R¹⁹and R²⁰ are independently a branched or straight chain C₁₋₁₀ alkanediyl;and, wherein X is independently C═O or SO₂; or, a salt, ester, hydrate,solvate, or stereoisomer thereof; and, a parenteral carrier systemcomprising one or more inactive pharmaceutical ingredients, wherein thepharmaceutical composition has a pH of 4.0-7.0 or 5.0-6.5.

Another aspect of the invention is a method for treating or preventinginjury to a biological material exposed to hypoxic or ischemicconditions comprising parenterally administering to the biologicalmaterial or a patient in need thereof a therapeutically effective doseof a compound according to the structure (III):

or a specific stereoisomer thereof according to the structure:

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are independently a side chain of anaturally-occurring or a non-naturally occurring amino acid (e.g.,CH₂CO₂H, (CH₂)₂CO₂H), a branched or straight chain C₁₋₁₀ alkyl, astraight or branched chain C₁₋₁₀ alkyl substituted with —ONO₂, or, astraight or branched chain C₁₋₁₀ alkyl substituted with —OH; wherein R¹⁹and R²⁰ are independently a branched or straight chain C₁₋₁₀ alkanediyl;and, wherein X is independently C═O or SO₂; or, a salt, ester, hydrate,solvate, or stereoisomer thereof.

In an exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, m is 3, Z is H, and R⁵and R⁶ are OH according to the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material. R³ and R⁴ are selectedfrom C(CH₃)₂, CH(CH₃), (CH₂)₂, and CH₂, Z is H, and, R⁵ and R⁶ areselected from NHCH₂COOH, NH(CH₂)₂COOH, NHCH₂ONO₂, NH(CH₂)₂ONO₂, andNH(CH₂)₂OH.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are SO₂, Z is H,R¹ and R² are CH₃, R³ and R⁴ are CH₂, and, R⁵ and R⁶ are OH according tothe free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, Z is H,R¹ and R² are CH₃, R³ and R⁴ are C(CH₃)₂, and, R⁵ and R⁶ are OHaccording to the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, Z is H,R¹ and R² are CH₃, R³ and R⁴ are (CH₂)₂, and, R⁵ and R⁶ are OH accordingto the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound, its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, R¹ andR² are CH₃, R³ and R⁴ are CH₂, Z is CH₃, and, R⁵ and R⁶ are OH accordingto the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, Z is H,R¹ and R² are CH₃, R³ and R⁴ are CH₂, and, R⁵ and R⁶ are NHCH₂CO₂Haccording to the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, Z is H,R¹ is CH₃, R² is CH₂CH₃, R³ and R⁴ are CH₂, and, R⁵ and R⁶ are OHaccording to the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X is C═0, Y is SO₂, Z isH, R¹ and R² are CH₃, R³ and R⁴ are CH₂, and, R⁵ and R⁶ are OH accordingto the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, Z is H,R¹ and R² are CH₃, R³ is CH₂, R⁴ is C(CH₃)₂, and, R⁵ and R⁶ are OHaccording to the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, Z is H,R¹ and R² are CH₃, R³ and R⁴ are CH₂, R⁵ is OH, and, R⁶ is NHCH₂CO₂Haccording to the free acid structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, X and Y are C═O, Z is H,R¹ and R² are CH₃, R³ and R⁴ are CH₂, R⁵ is OH, and, R⁶ is NH(CH₂)₂ONO₂according to the structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment of the compound (I), its pharmaceuticalcompositions, its methods of making and its methods of use for treatingor preventing injury to a biological material, m is 4, Z is H, and R⁵and R⁶ are OH according to the structure

or, a salt, ester, hydrate, or solvate thereof.

In another exemplary embodiment the pharmaceutical composition is madeby mixing the compound in a carrier of water; NaCl; a weak acid such asmono, di, or tricarboxylic acid (e.g., citric acid); and NaOH.

In another exemplary embodiment the pharmaceutical composition thecarrier is a sodium phosphate buffer of pH 4-7.

In another exemplary embodiment of the pharmaceutical composition, thecitric acid comprises in the range of 0.01% to 2% of the intravenouscarrier system.

In another exemplary embodiment of the pharmaceutical composition, thecitric acid comprises in the range 0.05% to 0.1% of the intravenouscarrier system.

In another exemplary embodiment of the pharmaceutical composition, thepH of the intravenous dosage form is in the range of 5.0-6.5 or 4.0-7.0.

In another exemplary embodiment of the pharmaceutical composition, theeffective dose is in the range of 0.1 to 100 mg/Kg, 0.1 to 200 mg/Kg, 5to 100 mg/Kg or 2 to 60 mg/Kg based on body weight.

In another exemplary embodiment, the pharmaceutical composition is aparenteral or other dosage form selected from intravenous, injection,infusion, continuous infusion, intradermal, intraarterial,intracerebral, intracerebroventricular, intracardiac, intraosseousinfusion, intralesional, intracranial, intraprostatical, intrapleural,intratracheal, intranasal, intravitreal, intravaginal, intrarectal,intratumoral, intramuscular, intraocular, intrathecal, subcutaneous,subconjunctival, transmucosal, intramuscular, intravesicular,intravesical, intracavernosal injection, intrapericardial,intraumbilical, intraocularal, absorption, adsorption, immersion,localized perfusion, intracisternal, bolus and epidural.

In another exemplary embodiment of the methods of treating according tothe invention, the compound is administered in a parenteral dosage formcomprising the compound, and, a parenteral carrier system or vehiclesolution comprising one or more inactive pharmaceutical ingredients.

In another exemplary embodiment of the methods of treating according tothe invention, the compound is administered intravenously in anintravenous dosage form, as a single bolus, as a repeat bolus or as acontinuous infusion.

In another exemplary embodiment of the methods of treating according tothe invention, m is 4, and the compound is administered in anintravenous dosage form comprising the compound and a parenteral carriersystem, which comprises one or more inactive pharmaceutical ingredients,accordingly.

In another exemplary embodiment of the methods of treating according tothe invention, the method further comprises continuously orintermittently administering the intravenous or other parenteral dosageform for a duration of 1 minute to 2 days or 1 to 5 hours.

In another exemplary embodiment of the methods of treating according tothe invention, the method further comprises administering apredetermined plurality of doses over a specified interval, for example,administering a dosing regimen of the intravenous dosage form, whereinthe dosing regimen comprises a predetermined plurality of doses over apredetermined duration at predetermined intervals.

In another exemplary embodiment of the methods of treating according tothe invention, the method further comprises administering a dosingregimen of the intravenous or other parenteral dosage form, wherein thedosing regimen comprises continuously administering a predetermined doseover a predetermined duration.

In another exemplary embodiment of the methods of treating according tothe invention, the hypoxic or ischemic condition results from an injuryto the biological material, the onset or progression of a disease whichadversely affects the biological material or hemorrhaging of thebiological material.

In another exemplary embodiment of the methods of treating according tothe invention, the biological material is contacted with the compoundbefore the injury, before the onset or progression of the disease, orbefore hemorrhaging of the biological material.

In another exemplary embodiment of the methods of treating according tothe invention, the injury is from an external physical source, such assurgery.

In another exemplary embodiment of the methods of treating according tothe invention, the hypoxic or ischemic conditions result in myocardialinfarction, sepsis, vascular abnormalities, cirrhosis, liver injury,kidney injury, vascular calcification, gastric injury induced by drugtreatment, burns, lung injury, neutrophil adhesion, leukocyte-mediatedinflammation, erectile dysfunction, irritable bowel syndrome,anti-nociceptive effects in post-inflammatory hypersensitivity, acutecoronary syndrome, cardiac arrest, planned cardiac bypass surgery,congestive heart failure, neonatal hypoxia/ischemia, myocardial ischemicreperfusion injury, unstable angina, post-angioplasty, aneurysm, trauma,stroke, hemorrhagic shock, and/or blood loss.

In an exemplary embodiment of the methods of making the pharmaceuticalcompositions of the compounds of the invention, the vehicle solution ismade by mixing 1 volume of 0.5N NaOH in saline (0.9% NaCl injectionsolution, Baxter), 4 volumes of 1% citric acid in sterile water, and 36volumes of saline. The pH of the resulting vehicle solution is about 6.

In another exemplary embodiment of the methods of making thepharmaceutical compositions of the compounds of the invention, thepredetermined concentration of the dosing solution is in the range of0.1 to 200 mg/mL, 1 to 100, or even 2 to 40 mg/mL, or even 5 to 6 mg/mLand the administered dose in the range of 0.1 to 200 mg/Kg, or even 2 to60 mg/Kg, or even specifically 20 mg/Kg, based on body weight.

In a further exemplary embodiment of the methods of making thepharmaceutical compositions of the invention, a predetermined volume ofdosing solution of bis-N-acetyl-cystine-trisulfide at a predeterminedconcentration between 2 and 40 mg/mL is prepared by weighing thecalculated amount of solid bis-N-acetyl-cystine-trisulfide into a clearglass vial and chilling the vial to −20° C. The required volume ofsolvent is prepared by diluting 0.011 mL of 0.5N NaOH in saline forevery 1 mg of bis-N-acetyl-cystine-trisulfide with a suitable volume ofabove vehicle solution such that the predetermined volume of dosingsolution is reached. The entire amount of solvent is mixed tohomogeneity, chilled to between 0° C. and 4° C. and added to the vial ofbis-N-acetyl-cystine-trisulfide. The resulting suspension is mixedimmediately until the solid is fully dissolved.

In one more exemplary embodiment of the methods of making thepharmaceutical compositions of the invention, a dosing solution ofbis-N-acetyl-cystine-trisulfide at 30 mg/mL is prepared by weighing thedesired amount of solid bis-N-acetyl-cystine-trisulfide into a clearglass vial and chilling the vial to −20° C. The solvent is prepared bychilling a suitable amount of a mixture of 1 volumes of 0.5N NaOH insaline and 2 volumes of above vehicle solution to between 0° C. and 4°C. One mL of this solvent is added to the vial per 30 mg ofbis-N-acetyl-cystine-trisulfide contained and the resulting suspensionmixed immediately until the solid is fully dissolved. This dosingsolution of bis-N-acetyl-cystine-trisulfide is stable for at least 1.5hours at room temperature and for at least 24 hours when frozen at −20°C. The dosing solution is warmed to room temperature prior tointravenous injection.

DESCRIPTION OF DRAWINGS OF EXEMPLARY EMBODIMENTS

FIG. 1 is a graph demonstrating the pharmacokinetics of biologicallyavailable blood sulfide measured after intravenous administration ofbis-N-acetyl-cystine-trisulfide at two doses (16 mg/kg and 32 mg/kg).Sulfide levels after IV bolus of vehicle are shown for comparison.Biologically available sulfide was measured by derivatization withmono-bromo-bimane (Wintner et al. 2010, Br. J. Pharmacology160:941-957). Sulfide was elevated 1 min after IV bolus administrationand decreased thereafter.

FIG. 2 is a graph demonstrating the pharmacokinetics of biologicallyavailable sulfide in blood as measured by derivatization withmono-bromo-bimane and of plasma thiosulfate after intravenousadministration of bis-N-acetyl-cystine-trisulfide as a 32 mg/kg boluscompared to administration of vehicle. Plasma thiosulfate was measuredby ion chromatography. Thiosulfate levels in blood paralleled bloodlevels of biologically available sulfide.

FIG. 3 is a graph demonstrating the pharmacokinetics of free sulfide inblood measured with a sulfide-specific electrode implanted in the venacava of rats receiving an IV bolus of 20 mg/kg bolus dose ofbis-N-acetyl-cystine-trisulfide or vehicle. Free sulfide levels weremeasured every second and are shown in minute intervals with breathingartifacts removed. No free sulfide was detectable.

FIG. 4 shows two bar graphs demonstrating the protection of the liversof mice from injury by dGal/LPS by an intraperitoneal (IP) bolus ofbis-N-acetyl-cystine-trisulfide of 20 mg/kg given 30 min after LPS,compared to vehicle administration. Liver injury was monitored by levelsof serum alanine transaminase (ALT), FIG. 4A, and serum aspartateaminotransferase (AST), FIG. 4B. Bis-N-acetyl-cystine-trisulfide reducedliver injury.

FIG. 5 is a bar graph demonstrating the protection of the livers of micefrom injury by dGal/LPS by an intraperitoneal bolus ofbis-N-acetyl-cystine-trisulfide of 40 and 60 mg/kg given 30 min afterLPS, compared to vehicle administration. Hepatic injury was determinedby measuring serum ALT. Bis-N-acetyl-cystine-trisulfide reduced liverinjury.

FIG. 6 is a graph demonstrating that biologically available bloodsulfide and plasma thiosulfate levels can be manipulated by repeated IVinjections to achieve elevated exposure levels. Increasedmono-bromo-bimane-reactive blood sulfide and blood thiosulfate levelspersisted for 3 hours after administration of two consecutive IV boli ofbis-N-acetyl-cystine-trisulfide at 0 and 15 min, compared toadministration of vehicle.

FIG. 7 is a graph demonstrating the protection of the livers of micefrom ischemia/reperfusion (I/R) injury by an intravenous bolus ofbis-N-acetyl-cystine-trisulfide at 5, 20 or 60 mg/kg, compared tountreated and vehicle-treated conditions. Hepatic injury was determinedby measuring plasma ALT.

FIG. 8 is a graph demonstrating the protection of the livers of micefrom ischemia/reperfusion (I/R) injury by an intravenous bolus ofbis-N-methylsulfonamide-cystine-trisulfide at 2, 5, or 20 mg/kg,compared to untreated and vehicle-treated conditions. Hepatic injury wasdetermined by measuring plasma ALT.

FIG. 9 is a graph demonstrating that blocking free thiols and perthiolsin rat blood with the thiol-reactive agent N-aminoethyl-maleimide (NAEM)reduces the generation of biologically available sulfide. Theconcentration of biologically available blood sulfide is shown on theleft y-axis, concentrations of bis-N-acetyl-cystine-trisulfide and NAEMare shown on the right y-axis relative to their initial concentrations.Time after bis-N-acetyl-cystine-trisulfide addition (sulfide andbis-N-acetyl-cystine-trisulfide curves) or NAEM addition (NAEM curve)shown on x-axis. Bis-N-acetyl-cystine-trisulfide and NAEM were measuredin blood by LC/MS/MS, biologically available sulfide by derivatizationwith mono-bromo-bimane.

FIGS. 10A, 10B, and 10C are synthetic reactions according to Scheme 1for preparation of embodiments of the present invention.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

In the following description, details are set forth to provide a morethorough and detailed description of various non-limiting exemplaryembodiments.

The term “comprise” (and variations thereof) is to be construed as open,non-limiting, and open to the inclusion of other components,ingredients, steps or acts, and the like.

The terms “biological material” (and variations thereof) means mammalian(such as human) cells, tissues and organs, whereby it may be disposedwithin the organism, removed/isolated from the organism, or, on thewhole of the organism.

The term “cell” (and variations thereof) refers to mammalian cells, suchas from a human, monkey, mouse, rat, rabbit, hamster, goat, pig, dog,cat, ferret, cow, sheep, horse or the like. The cells may be diploid orhaploid (i.e., sex cells). The cells may also be polyploid, aneuploid,or anucleate. The cell may be from a particular tissue or organ, such asheart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein,artery, cornea, blood, small intestine, large intestine, brain, spinalcord, smooth muscle, skeletal muscle, ovary, testis, uterus, umbilicalcord or the like. The cell may also be a platelet, myelocyte,erythrocyte, lymphocyte, adipocyte, fibroblast, epithelial cell,endothelial cell, smooth muscle cell, heart muscle, skeletal musclecell, endocrine cell, glial cell, neuron, secretory cell, barrierfunction cell, contractile cell, absorptive cell, mucosal cell, limbuscell (from cornea), stem cell (totipotent, pluripotent or multipotent),unfertilized or fertilized oocyte, sperm or the like.

The terms “tissue” and “organ” (and variations thereof) whereby “tissue”refers to an aggregate of similar cells forming a definite kind ofstructural material, and whereby “organ” refers to a particular type oftissue. The tissue or organ may be “isolated” from the subject orpatient meaning that it is not located within the subject or patient orhas been removed such.

The terms “hypoxia” and “hypoxic” (and variations thereof) refer to anenvironment having reduced dissolved oxygen content of a body of waterdetrimental to aerobic organisms or a pathological condition in whichthe body as a whole or region of the body is deprived of adequate oxygensupply. Hypoxia occurs when the normal physiologic levels of oxygen arenot supplied to a cell, tissue, or organ.

The term “normoxia” (and variations thereof) refers to normalphysiologic levels of oxygen for the particular cell type, cell state ortissue in question.

The term “anoxia” (and variations thereof) refers the absence of oxygen.

The term “hypoxic conditions” (and variations thereof) refer toconditions leading to cellular, organ or organismal hypoxia. Hypoxicconditions include conditions where the concentration of oxygen is at orless than normal atmospheric conditions. For example, the concentrationof oxygen may be less than about any value in the range of 20.8% to0.5%, or trace to undetectable concentrations, whereby the percentagemay be that at 1 atmosphere of pressure (101.3 kPa).

The term “ischemia/reperfusion injury” denotes damage to tissues,vasculature, organs or body parts that is caused by the return of bloodflow after a time period of oxygen deprivation resulting from ischemia.Reperfusion injury is caused both by the return of oxygen to ischemictissues and by the inflammatory response solicited by tissues damaged bythe return of circulation after the ischemic period. Return of bloodflow exposes cells to oxygen which causes intracellular radicalgeneration and damage to cellular proteins, DNA, and the plasmamembrane. This can lead to calcium overload, energy failure and necrosisor the induction of apoptosis. In reperfusion injury, leukocytes (whiteblood cells) entering tissues and organs from the circulation releaseinflammatory mediators such as cytokines and interleukins, proteaseenzymes and free radicals that further damage the tissue or organ.Injured endothelium recruits leukocytes and platelets which can lead toobstruction of capillaries and further ischemia.

The term “ischemia” is a condition in which the blood flow (and oxygen)is restricted to a part of the body. Hypoxic or ischemic conditions mayresult from any one of the following: an injury to the biologicalmaterial; the onset or progression of a disease that adversely affectsthe biological material; or, cardiac ischemia or hemorrhaging of thebiological material. The biological material may be treated with thecompounds of the invention (i.e., API) before the injury; before theonset or progression of the disease; before hemorrhaging of thebiological material, or the like. The injury may be from an externalphysical force, such as surgery.

The terms “biologically available sulfide” (and variations thereof)refer to molecules detectable by derivatization with the chemicalmono-bromo-bimane and measurement of the subsequently producedsulfide-di-bimane.

The terms “free sulfides” (and variations thereof) refer to sulfidesthat exist in an ionized or un-ionized state and are unbound andunsequestered by any protein, peptide or other molecule in thebloodstream. Free sulfides can be detected by the presence of exhaledH2S on an animal or patient's breath or by a membrane-mediatedelectrochemical probe which specifically measures unbound H2S moleculesin solution. Free sulfide in the bloodstream can cause unwanted sideeffects.

The terms “inactive pharmaceutical ingredient” (and variations thereof)refer to any component of a drug product other than the activeingredient. (See 21 CFR 210.3(b)(8)).

The terms “active pharmaceutical ingredient” or “API” refer to anycomponent of a drug product intended to furnish pharmacological activityor other direct effect in the diagnosis, cure, mitigation, treatment, orprevention of disease, or to affect the structure or any function of thebody of humans or other animals. (See 21 CFR 210.3(b)(7)). APIs includethose components of the product that may undergo chemical change duringthe manufacture of the drug product and be present in the drug productin a modified form intended to furnish the specified activity or effect.The polysulfide compounds, both linear and cyclic as well as symmetricand asymmetric, disclosed herein are APIs.

The term “dosage form” indicates a packaged form of the API suitable foradministration as a medication or drug.

A “pharmaceutical composition” refers to a formulation of a compound(i.e., an API) of the invention and a medium generally accepted in theart for the delivery of the biologically active compound to mammals,e.g., humans. Such a medium (e.g., vehicle solution) includes allpharmaceutically acceptable carriers, diluents or excipients (i.e.,inactive pharmaceutical ingredients) therefore. As used herein, theterms “vehicle solution” and “carrier system” (and variations of both)are used interchangeably.

The terms “complexing agent” refer to a molecule, typically an organicmolecule, which binds a metal ion through two or more of the complexingagent's atoms.

The term “co-solvent” refers to any solvent or compound present in amixture in addition to the primary solvent. Co-solvents are typicallyadded to increase or decrease the solubility of the solutes.

The term “stereoisomer” refers to a compound made up of the same atomsbonded by the same bonds but having different three-dimensionalstructures, which are not interchangeable. The present inventioncontemplates various stereoisomers and mixtures thereof and includes“enantiomers”, which refers to two stereoisomers whose molecules arenonsuperimposeable mirror images of one another, and diastereomers,which are stereoisomers of compounds with more than one chiral centerthat are not enantiomers.

The compounds of the invention, or their pharmaceutically acceptablesalts, may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R) or (S) or, as(D) or (L) for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Therefore, it is to be understood that where the structuralrepresentations of the inventive compounds set forth herein are shown asstereospecific it is for illustrative purposes only and the invention isnot intended to be so limited.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Ultra Version 11.0software naming program (CambridgeSoft).

The term “alkyl” refers to a straight or branched chain hydrocarbonmoiety. In one embodiment, the alkyl is C_(n)H_(2n+1) where n is 1-10.

The term “alkanediyl” refers to a straight or branched chain divalenthydrocarbon moiety. In one embodiment, the alkanediyl is C_(n)H_(2n)where n is 1-10.

The term “salts” (and variations thereof) of the instant compounds referto pharmaceutically suitable (i.e., pharmaceutically acceptable) saltsincluding, but not limited to, acid addition salts formed by mixing asolution of the instant compound with a solution of a pharmaceuticallyacceptable acid. The pharmaceutically acceptable acid may behydrochloric acid, methanesulphonic acid, fumaric acid, maleic acid,succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid,tartaric acid, carbonic acid or phosphoric acid. Variouspharmaceutically acceptable salts are well known in the art and may beused with the instant compound such as those disclosed in Berge S M etal., “Pharmaceutical Salts.” J. Pharm. Sci. 66:1-19 (1977) and Haynes DA et al., “Occurrence of pharmaceutically acceptable anions and cationsin the Cambridge Structural Database,” J. Pharm. Sci. 94:2111-2120(2005), which are hereby incorporated herein by reference. For example,the list of FDA-approved commercially marketed salts includes acetate,benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calciumedetate, camsylate, carbonate, chloride, citrate, dihydrochloride,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, mitrate,pamoate, pantothenate, phosphate, diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,teoclate, and triethiodide.

The term “hydrates” (and variations thereof) of the instant compoundsrefer to pharmaceutically suitable (i.e., pharmaceutically acceptable)hydrates being compounds formed by the addition of water or its elementsto a host molecule (e.g., the free form version of the compound)including, but not limited to, monohydrates, dihydrates, etc.

The term “solvates” (and variations thereof) of the instant compoundsrefer to pharmaceutically suitable (i.e., pharmaceutically acceptable)solvates, whereby solvation is an interaction of a solute with thesolvent which leads to stabilization of the solute species in thesolution, and whereby the solvated state is an ion in a solutioncomplexed by solvent molecules.

The invention disclosed herein is also meant to encompass allpharmaceutically acceptable versions of the compounds of the inventionherein being isotopically-labelled by having one or more atoms replacedby an atom having a different atomic mass or mass number. Examples ofisotopes that can be incorporated into the disclosed compounds includeisotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine,chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O,¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. Thesecompounds could be useful to help determine or measure the effectivenessof the compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled versions of the compounds of theinvention, for example, those incorporating a radioactive isotope, areuseful in drug and/or substrate tissue distribution studies. Theradioactive isotopes tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, areparticularly useful for this purpose in view of their ease ofincorporation and ready means of detection.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. As used herein, “stable” refers to theconcentration of the active polysulfide composition, the pH of thepolysulfide composition and/or any polysulfide degradation productsremaining within a specified range.

Pharmaceutical compositions of the invention are administered byinjection or infusion (e.g. intravenous, subcutaneous, etc.) andinclude, but are not limited to, liposomal injectables or a lipidbilayer vesicle having phospholipids that encapsulate an active drugsubstance. Injection includes a sterile preparation intended forparenteral use.

Administration of the compounds of the invention, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of administration of agents for serving similar utilities. Thepharmaceutical compositions of the invention can be prepared bycombining a compound of the invention with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated. The terms “parenteral carrier system” (including variationsthereof such as the various specific injectable and infusible dosageforms) refer to compositions comprising one or more pharmaceuticallysuitable excipients, such as solvents (e.g. water) and co-solvents,solubilizing agents, wetting agents, suspending agents, thickeningagents, emulsifying agents, chelating agents, buffers, pH adjusters,antioxidants, reducing agents, antimicrobial preservatives, bulkingagents, protectants, tonicity adjusters, and special additives.

The term “parenteral” as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques. Pharmaceutical compositions of the invention are formulatedso as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.

Five distinct classes of injectable dosage forms exist as defined by theUSP: emulsions, lipids, powders, solutions and suspensions. Emulsioninjection includes an emulsion comprising a sterile, pyrogen-freepreparation intended to be administered parenterally. Lipid complex andpowder for solution injection are sterile preparations intended forreconstitution to form a solution for parenteral use.

Powder for suspension injection is a sterile preparation intended forreconstitution to form a suspension for parenteral use. Powderlyophilized for liposomal suspension injection is a sterile freeze driedpreparation intended for reconstitution for parenteral use that isformulated in a manner allowing incorporation of liposomes, such as alipid bilayer vesicle having phospholipids used to encapsulate an activedrug substance within a lipid bilayer or in an aqueous space, wherebythe formulation may be formed upon reconstitution.

Powder lyophilized for solution injection is a dosage form intended forthe solution prepared by lyophilization (“freeze drying”), whereby theprocess involves removing water from products in a frozen state atextremely low pressures, and whereby subsequent addition of liquidcreates a solution that conforms in all respects to the requirements forinjections. Powder lyophilized for suspension injection is a liquidpreparation intended for parenteral use that contains solids suspendedin a suitable fluid medium, and it conforms in all respects to therequirements for sterile suspensions, whereby the medicinal agentsintended for the suspension are prepared by lyophilization.

Solution injection involves a liquid preparation containing one or moredrug substances dissolved in a suitable solvent or mixture of mutuallymiscible solvents that is suitable for injection. Solution concentrateinjection involves a sterile preparation for parenteral use that, uponaddition of suitable solvents, yields a solution conforming in allrespects to the requirements for injections.

Suspension injection involves a liquid preparation (suitable forinjection) containing insoluble solid and/or immiscible liquid particlesdispersed throughout a liquid phase. Suspension liposomal injection is aliquid preparation (suitable for injection) having an oil phasedispersed throughout an aqueous phase in such a manner that liposomes (alipid bilayer vesicle usually containing phospholipids used toencapsulate an active drug substance either within a lipid bilayer or inan aqueous space) are formed. Suspension sonicated injection is a liquidpreparation (suitable for injection) containing solid particlesdispersed throughout a liquid phase, whereby the particles areinsoluble. In addition, the product may be sonicated as a gas is bubbledthrough the suspension resulting in the formation of microspheres by thesolid particles.

The parenteral carrier system includes one or more pharmaceuticallysuitable (i.e., USP and FDA-approved) excipients and/or diluents, suchas solvents and co-solvents, solubilizing agents, wetting agents,suspending agents, thickening agents, emulsifying agents, chelatingagents, buffers, pH adjusters, antioxidants, reducing agents,antimicrobial preservatives or other preservatives, dispersing agents,surfactant, bulking agents, protectants, tonicity adjusters,emulsifiers, stabilizers, glidants, isotonic agents, and specialadditives.

The terms “therapeutically effective dose” (and variations thereof)refer to an amount, dose or dosing regimen of a compound (i.e., activepharmaceutical ingredient, prodrug or precursor thereof) that uponinteraction with a biological material is sufficient to treat or preventinjury of a biological material (e.g., induce a measurable result)exposed to hypoxic or ischemic conditions, whereby such dose may varydepending on the form of the compound, the biological material'scondition and/or severity, the route of administration, the age of thebiological material, and the like.

“Therapeutically effective dose” may also mean a dose administered to ahuman subject/patient in a controlled Phase II or Phase III clinicaltrial that causes a statistically significant benefit on a predefinedclinical endpoint (e.g., mortality). A therapeutically effective dosemay also be a dose that enhances the survivability of biological matterin response to a disease or injury or an amount that induces stasis orpre-stasis in the biological matter.

The instant dosage form and routes of administration of a pharmaceuticalcomposition include parenteral by injection or infusion (or otherparenteral) such as intravenous, injection, infusion, continuousinfusion, intradermal, intraarterial, intracerebral,intracerebroventricular, intracardiac, intraosseous infusion,intralesional, intracranial, intraprostatical, intrapleural,intratracheal, intranasal, intravitreal, intravaginal, intrarectal,intratumoral, intramuscular, intraocular, intrathecal, subcutaneous,subconjunctival, transmucosal, intramuscular, intravesicular,intravesical, intracavernosal injection, intrapericardial,intraumbilical, intraocularal, absorption, adsorption, immersion,localized perfusion, intracisternal, bolus and epidural.

The compounds of the invention may be administered to the biologicalmaterial in a dose and for a duration sufficient to protect thebiological material from one or more of the following: damage or deathresulting from the injury; onset or progression of disease; hemorrhagingin the biological material, or the like.

The mammalian biological material may be one or more of the following:cells, tissues, organs, organisms or an animal. The mammal may be ahuman. The biological material may be platelets. The biological materialmay be used for transplantation. The biological material may be at riskof reperfusion injury. The biological material may also be at risk ofhemorrhagic shock.

The hypoxic or ischemic conditions may result in one or more of thefollowing: myocardial infarction, sepsis, vascular abnormalities,cirrhosis, liver injury, kidney injury, vascular calcification, gastricinjury induced by drug treatment, burns, lung injury, neutrophiladhesion, leukocyte-mediated inflammation, erectile dysfunction,irritable bowel syndrome, anti-nociceptive effects in post-inflammatoryhypersensitivity, acute coronary syndrome, cardiac arrest, plannedcardiac bypass surgery, congestive heart failure, neonatalhypoxia/ischemia, myocardial ischemic reperfusion injury, unstableangina, post-angioplasty, aneurysm, trauma, stroke, hemorrhagic shock,and/or blood loss, or the like.

The compounds of the invention may be used to treat or prophylacticallytreat various diseases and disorders such as those disclosed in U.S.Patent Application Publication No. 2008/0199541. Other such treatableconditions include myocardial infarction, sepsis (Hui, et al. J Infect(2003) 47:155-160), congestive heart failure, vascular abnormalities incirrhosis (Fiorucci S, et al., Hepatology (2005) 42:539-548), as acardioprotectent (Geng, et al., Biochem Biophys Res Commun (2004)313:362-368), as a neuroprotectant (Qu K. et al, Stroke (2006)37:889-893), myocardial ischemia reperfusion injury (Johansen et al.,Basic Res Cardiol (2006) 101:53-60), vascular calcification (Wu et al.,Acta Pharmacol Sin. (2006) 27:299-306), gastric injury induced by drugtreatment (Fiorucci, S. et al., Gastroenterology (2005) 129:1210-1224),neutrophil adhesion and modulation of leukocyte-mediated inflammation(Zanardo et al., FASEB J. (2006) 20:2118-2120), erectile dysfunction(Srilatha B. et al., Eur J. Pharmacol. (2006) 535:280-282), irritablebowel syndrome (Distrutti E., et al., JPET (2006) 319:447-458), and,anti-nociceptive effects in post-inflammatory hypersensitivity (ibid.).

The compounds of the invention may also be used to treat orprophylactically treat to prevent injury to biological matter exposed toischemic or hypoxic conditions. The compounds of the invention may beused to treat patients that have undergone, are undergoing, or, aresusceptible to injury, trauma or critical care treatment. Injury may becaused by external insults, such as burns, wounds, amputations, gunshotwounds, surgical trauma, abdominal surgery, prostate surgery, limbsurgery, internal insults (such as septic shock), stroke or cardiacarrest, heart attack that result in the acute reduction in circulation,reductions in circulation due to non-invasive stress (such as exposureto cold or radiation) or the like. At the cellular level, injury mayresult in exposure of cells, tissues and/or organs to hypoxia therebyresulting in induction of programmed cell death, or apoptosis.

The compounds of the invention may also be used for treating reperfusioninjury such as edema through vascular leak and acute inflammation causedby penetration of activated leukocytes into tissue and cell death bynecrosis and apoptosis, as well as other etiologies. Reperfusion injurycan occur following myocardial reperfusion after an acute myocardialinfarction, stroke, cardiac arrest, or coronary artery bypass graft(CABG) surgery. Reperfusion injury is noted following thetransplantation of an organ or following resuscitation after hemorrhagicshock or severe bleeding in traumatized patients.

The compounds of the invention are also useful for preventing ortreatment of hypoxic or ischemic injury related to transplantation of atissue or an organ. The compounds of the invention are also useful inthe prevention or treatment of delayed graft function.

The compounds of the invention are also useful for inducing tissueregeneration and wound healing by prevention/delay of biologicalprocesses that may result in delayed wound healing and tissueregeneration. In addition to wound healing, methods can be implementedto prevent or treat trauma such as cardiac arrest or stroke, andhemorrhagic shock. The compounds of the invention are also useful forreducing the risk of trauma from emergency surgical procedures, such asthoracotomy, laparotomy, and splenic transaction or cardiac surgery,aneurysm, surgery, brain surgery and the like. The compounds of theinvention may also be used to prevent or treat injury resulting fromSystemic Inflammatory Response Syndrome (SIRS), Acute RespiratoryDistress Syndrome (ARDS), kidney failure, liver failure and multi-organfailure.

The compounds of the invention are also useful for methods of enhancingsurvivability and prevent ischemic injury resulting from cardiac arrestor stroke by providing a therapeutically effective amount of thecomposition to the patient before, after, or both before and aftermyocardial infarction, cardiac arrest or stroke.

The compounds of the invention may also be used to treat or preventischemia/reperfusion injury; an inflammatory disease or disorder; or, avascular leak.

The compounds of the invention are also useful in methods ofpre-treating a biological material, e.g., a patient, prior to anischemic or hypoxic injury or disease insult. These methods can be usedwhen an injury or disease with the potential to cause ischemia orhypoxia is scheduled or elected in advance, or predicted in advance tolikely occur. Examples include, but are not limited to, major surgerywhere blood loss may occur spontaneously or as a result of a procedure,cardiopulmonary bypass in which oxygenation of the blood may becompromised or in which vascular delivery of blood may be reduced (as inthe setting of coronary artery bypass graft (CABG) surgery), or in thetreatment of organ donors prior to removal of donor organs for transportand transplantation into a recipient in need of an organ transplant.Examples include, but are not limited to, medical conditions in which arisk of injury or disease progression is inherent (e.g., in the contextof unstable angina, following angioplasty, bleeding aneurysms,hemorrhagic strokes, following major trauma, hemorrhaging or bloodloss), or in which the risk can be diagnosed using a medical diagnostictest.

The compounds of the invention may be formulated into various dosageforms for various routes of administration for treating tissues, organs,limbs and even whole organisms with a therapeutically effective dose ofcompound (i.e., API) to protect such from the detrimental effects ofinjury. Where medical attention is not readily available, administrationof the compounds of the invention provides time for the patient untilthey can receive other medical attention.

The compounds of the invention are also useful for inducing tissueregeneration and wound healing by preventing and/or delaying biologicalprocesses to promote wound healing and tissue regeneration. Where thereis a substantial wound to the limb or organism, treating the biologicalmatter with the compounds of the invention aid in the wound healing andtissue regeneration process by managing the biological processes thatinhibit healing and regeneration. Other methods of using the compoundsof the invention includes preventing or treating trauma such as cardiacarrest or stroke, and hemorrhagic shock. The risk of trauma fromemergency surgical procedures, such as thoracotomy, laparotomy, andsplenic transaction or cardiac surgery, aneurysm, surgery, brain surgeryand the like are also treatable using the compounds of the invention.

Other methods of using the compounds of the invention include enhancingsurvivability and preventing ischemic injury resulting from cardiacarrest or stroke and reducing ischemic injury in a patient sufferingfrom or at risk of cardiac arrest or stroke. A therapeutically effectivedose of the compounds of the invention are administered to the patientbefore, after, or both before and after myocardial infarction, cardiacarrest or stroke.

Other methods include pretreating the patient's biological materialprior to an ischemic or hypoxic injury or disease insult. Such methodsare used when an injury or disease, with the potential to cause ischemiaor hypoxia, is scheduled or elected in advance, or predicted in advanceto likely occur, such as major surgery where blood loss may occurspontaneously or as a result of a procedure. Other anticipatedprocedures include cardiopulmonary bypass in which oxygenation of theblood may be compromised or vascular delivery of blood may be reduced(as in the setting of coronary artery bypass graft (CABG) surgery).Still other such procedures include treatment of organ donors prior toremoval of donor organs for transport and transplantation into arecipient in need of an organ transplant. Other medical conditionsinclude a risk of injury or disease progression is inherent, such as inthe context of unstable angina, following angioplasty, bleedinganeurysms, hemorrhagic strokes, following major trauma or blood loss, orcongestive heart failure. The risk may be capable of being diagnosedusing a medical diagnostic test.

The compounds of the invention may also be used for enhancingsurvivability and preventing irreversible tissue damage from blood lossor other lack of oxygenation to cells or tissue, such as from lack of anadequate blood supply. Such tissue damage may result from actual bloodloss or from conditions or diseases that cause blockage of blood flow tocells or tissue. Reduced blood pressure locally or overall in anorganism may also occur, which reduces the amount of oxygen that iscarried in the blood, and/or which reduces the number of oxygen carryingcells in the blood. Such conditions and/or diseases include blood clotsand embolisms, cysts, growths, tumors, anemia, sickle cell anemia,hemophilia, other blood clotting diseases such as von Willebrand andITP, atherosclerosis and the like. Such conditions and diseases alsoinclude those that create essentially hypoxic or anoxic conditions forcells or tissue in an organism because of an injury, disease, orcondition.

The compounds of the invention may also be used to enhance thesurvivability of and prevent injury or damage to biological materialundergoing hemorrhagic shock. The method includes treating thebiological material at risk of or in a state of hemorrhagic shock with atherapeutically effective dose of the API within one hour of the injuryor sooner. The patient may be transported to a controlled environment,such as surgery, where the initial cause of the injury can be addressed.The patient can then be brought back to normal function in a controlledmanner. The first hour after injury, referred to as the golden hour, iscritical to a successful outcome.

The compounds of the invention may also be used in the treatment ofneurodegenerative diseases associated with ischemia, hypoxia,hypothermia, hyperproliferative disorders, immune disorders and thelike. The biological condition may include one or more of neurologicaldisease, cardiovascular disease, metabolic disease, infectious disease,lung disease, genetic disease, autoimmune disease, immune-relateddisease and the like.

The compounds of the invention may also be used to enhance thesurvivability of ex vivo biological matter (such as isolated cells,tissues and organs) subjected to hypoxic or ischemic conditions. Othersuch ex vivo biological material include platelets and other bloodproducts as well as tissues and organs to be transplanted.

The compounds of the invention may also be used to enhance survivabilityof biological material in the laboratory or research context, such aswhere cell lines or laboratory organisms are purposefully subjected tohypoxic or ischemic conditions, such as during cryopreservation andstorage. Cells, tissues or organs may also be stored or transported inthe presence of the compounds of the invention.

The compounds of the invention may also be used to increase thesurvivability of donor tissues and organs extending the time before thedonor tissue is transplanted into a recipient and blood flow isrestored. Such methods may be combined with known preservation methodsand materials, such as preservation agents and oxygen perfusion. Methodsof using the compounds of the invention provide a way of enhancingsurvivability of platelets stored in an anoxic environment by treatingthe platelets with a therapeutically effective dose of the compounds ofthe invention during storage.

The compounds of the invention are also useful for preserving bothnon-living biological material and preserving or extending theshelf-life of non-biological material by treating the non-livingbiological matter or non-biological material with a therapeuticallyeffective dose of the API.

The chemical name of a particular API compound is(R,R′)-3,3′-trithiobis-2-acetamidopropanoic acid,bis-N-acetyl-cystine-trisulfide, or S,S′-di-N-acetyl-cysteine-sulfide,and it has the following free acid form structure:

The chemical name of another particular API compound is(R,R′)-3,3′-trithiobis-2-(methylsulfonylamido)propanoic acid orbis-N-methylsulfonyl-cystine-trisulfide and it has the following freeacid form structure:

In addition to the free acid form, these API compounds may also existand be pharmaceutically active as a salt, ester, a hydrate, or asolvate. Their metabolites are also pharmacologically active. By meansof illustration, bis-N-acetyl-cystine-trisulfide is a double-cappedpolysulfide whose capping moiety consists of N-acetyl-cysteine groups.These capping groups have the benefit of low toxicity to biologicalmaterials in their reduced sulfide state. N-acetyl-cysteine has anintravenous LD₅₀ many mammals of approximately 300 mg/kg or greater.Specifically, N-acetyl-cysteine demonstrated an LD₅₀ IV in mouse of3,800 mg/kg, an LD₅₀ IV in rat of 1,140 mg/kg; and an LD₅₀ IV in dog of700 mg/kg.

The therapeutically effective dose of the API may be any amount in therange of 1 ppm, mg, mg/kg, or mg/m² to 1000 ppm, mg, mg/kg, or mg/m² orany range of amounts thereof. Alternatively, the dose or amount may beexpressed as mM or M.

In various exemplary embodiments, a biological material is exposed tothe compounds of the invention for any duration up to 30 seconds, 30minutes, 1 hour, 1 day, 1 week, or any duration or range thereof.

Exemplary intravenous rates are in the range of 1 gtts/min or μgtts/minto 100 gtts/min or μgtts/min or any value or range thereof. Thetherapeutically effective dose may also be in terms of solution volumeand API concentration, whereby the volume may be any value or range inthe range of 1 ml to 1000 ml.

Methods of preparing parenteral dosage forms are set forth in Remington:The Science and Practice of Pharmacy, 21st Edition (Philadelphia Collegeof Pharmacy and Science, 2005).

The therapeutically amount/dose of the API administered to biologicalmaterial can be about, at least, at least about, or at most about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg, mg/kg,or mg/m2, or any range derivable therein.

Alternatively, the therapeutically effective amount/dose of the API maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000μM, mM or M, or any range derivable therein.

The biological material may be exposed to the instant parenteral dosageform containing the API and the parenteral carrier system for about, atleast, at least about, or at most about 30 seconds, 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2,3, 4, 5, 6, 7 days or more, any range or combination therein, or in anysuitable continuous or intermittent (or combinations thereof) dosingregimen.

Where administration is intravenous, the dosage form may be administeredat a flow rate of about, at least about, or at most about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 gtts/min or μgtts/min, or any range derivable therein.

The amount/duration of time may be about, at least about, or at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7days, 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12months, or any range derivable therein.

The amount of the parenteral solution may also be specified by volume(depending on the concentration of the API). The volumes may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mL or L, orany range therein, which may be administered as the total dosingregimen, in a single session, or, in a defined set of sessions.

EXAMPLES Example 1 Preparation of Linear Symmetrical or AsymmetricalTrisulfides

The following reaction schemes illustrate methods of synthesizing therepresentative compounds of the present invention. It is understood thatone skilled in the art may be able to make these compounds by similarmethods or by combining other methods known to one skilled in the art.It is also understood that one skilled in the art would be able to make,in a similar manner as described below, other compounds of the presentinvention not specifically illustrated below by using the appropriatestarting components and modifying the parameters of the synthesis asneeded. In general, starting components may be obtained from commercialsources such as Aldrich Chemical Co. (Milwaukee, Wis., USA), etc. orsynthesized according to sources known to those skilled in the art (see,e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,5^(th) edition (Wiley, December 2000) or prepared as described in thisinvention.

Reaction Scheme 1 shown in FIGS. 10A, 10B, and 10C illustrates asynthetic approach to prepare both symmetrical and asymmetrical lineartrisulfides, e.g., compounds of structure (I) wherein m is 3. The keyintermediate,(R)-2-acetamido-3-((1,3-dioxoisoindolin-2-yl)disulfanyl)propanoic acid,was prepared by reacting of N-acetyl-L-cysteine with equal molar of2,2′-thiodiisoindoline-1,3-dione in 80% CH₃CN/H₂O at room temperaturefor 24 hours. This pure intermediate was obtained by chromatography onsilica-gel, and characterized by ¹H-NMR and LC-MS. Reaction of(R)-2-acetamido-3-((1,3-dioxoisoindolin-2-yl)disulfanyl)propanoic acidwith the corresponding protected cysteine- or thio-containing analogs in50% i-PrOH/H₂O at room temperature gave its corresponding desiredproducts. Final pure products, e.g.,(R,R′)-3,3′-trithiobis-2-acetamidopropanoic acid (1-a), were whitesolids and all of these pure products were obtained by preparativehigh-performance liquid chromatography (HPLC), and characterized by¹H-NMR, analytical HPLC, LC-MS and MS. The non-limiting examplesdisclosed in Reaction Scheme 1 have (M+H)⁺ by positive APCI-MS: 357.00(I-a); 414.10 (I-h); 371.10 (I-g); 385.07 (I-c); 393.06 (I-j).

Reaction Scheme 2 below further illustrates the preparation of thesymmetrical compound(R,R′)-3,3′-trithiobis-2-(methylsulfonylamido)propanoic acid (I-d). Thissynthetic scheme could be modified to prepare other symmetrical andasymmetrical compounds of structure (I). The key intermediate,(R)-2-methylsulfonylamido-3-((1,3-dioxoisoindolin-2-yl)disulfanyl)propanoicacid, was prepared by reacting N-methylsulfonyl-L-cysteine with equalmolar of 2,2′-thiodiisoindoline-1,3-dione in 80% CH₃CN/H₂O at roomtemperature for 24 hours. This pure intermediate was obtained bychromatography on silica-gel, and characterized by ¹H-NMR and LC-MS.Reaction of(R)-2-methylsulfonyl-3-((1,3-dioxoisoindolin-2-yl)disulfanyl)propanoicacid with the corresponding protected cysteine- or thio-containinganalogs in 50% i-PrOH/H₂O at room temperature gave its correspondingdesired products. The pure acid form of this compound (1-d), a whitesolid, was obtained by purification using preparative high-performanceliquid chromatography (HPLC), and it was characterized by ¹H-NMR,analytical HPLC, LC-MS and MS. It has 429.01 as (M+H)⁺ by positiveAPCI-MS.

As shown in Reaction Scheme 2 above, the N-methylsulfonyl-L-cysteine canbe prepared by reacting the compound represented by the structure

with methanesulfonyl chloride to produce a reaction product representedby the structure

and treating the reaction product with triethylsilane and acid (such astrifluoroacetic acid) to produce N-methylsulfonyl-L-cysteine.

Reaction Scheme 2 illustrates preparation of a specific compound of theinvention. However, as a general process for preparation of a variety ofcompounds according to the invention, the reaction can be illustrated asfollows:

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R⁵ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein Z isselected from H, CH₃, and CH₂CH₃. The((1,3-dioxoisoindolin-2-yl)disulfanyl)-containing intermediate isprepared in acetonitrile and water, isolated, and reacted with thecysteine or thio-containing analog in 50% alcohol and water to producethe final product as described above.

(R,R′)-3,3′-trithiobis-2-acetamidopropanoic acid, also namedN,N′-diacetyl-L-cystine trisulfide or S,S′-di-N-acetyl-cysteine-Sulfide,can also be prepared as shown in Reaction Scheme 3 (WO9948865/U.S. Pat.No. 6,288,250): N,N′-thio-diphthalimide (11 mmol) was added to asolution of N-Acetyl-L-cysteine (23 mmol) in isopropanol. The reactionmixture was stirred at room temperature for 24 hours yielding a clearsolution. After evaporation of isopropanol, the resulting whiteprecipitate was removed by filtration. The filtrates were lyophilized togive the crude product. The pure trisulfide as a white solid wasobtained by HPLC purification.

The compounds of the present invention may also be made by the followingsynthetic scheme disclosed in Mott A W et al. (Synthesis, (1984), (8),657-60). A general synthetic scheme is shown in Reaction Scheme 4.

Suitable sulfur-transferring coupling agents may be used such as2,2′-thiodiisoindoline-1,3-dione, N,N′-dibenzimidazyl sulfide,thiobis(imidazole), sulfur dichloride, sulfur monochloride, or elementalsulfur.

Example 2 Preparation of Cyclic Trisulfides

Compounds of structure (II) or (III) can be prepared by a modificationof the procedure described by Lundin et al (Tetrahedron Letters 1994,35:6339-6342) to favor the intramolecular oxidation of the lineardisulfide starting compound. For example,(5R,14S,17R)-17-acetamido-14-(carboxymethyl)-7,10,13,16-tetraoxo-1,2,3-trithia-6,9,12,15-tetraazacyclooctadecane-5-carboxylicacid was prepared as shown in Reaction Scheme 5: The starting material,Ac-Cys-Asp-Gly-Gly-Cys-OH, was prepared by FMOC-strategy on acid-labileresin supports using a commercially available peptide synthesizer, andwas purified by reverse-phase HPLC using a CH₃CN/H₂O (0.1% TFA)gradient. To a dilute solution (0.07 mM) of this linear peptide in 80%CH₃CN/H₂O was added 1 eq. of 2,2′-thiodiisoindoline-1,3-dione, and thisreaction mixture was stirred under argon at room temperature for 24hours. The desired cyclic trisulfide was obtained by reverse-phase HPLCpurification using a CH₃CN/H₂O (0.1% TFA) gradient, and characterized by¹H-NMR, analytical HPLC, LC-MS and MS. It is a white powder which has526.64 as (M+H)⁴ by positive APCI-MS.

Example 3 Dosing Solution Preparation

Materials: Bis-N-acetyl-cystine-trisulfide cystine-trisulfide was storedat −20° C. Solution A was 1% citric acid in sterile water. Solution Bwas 0.5 N NaOH in saline (0.9% NaCl injection solution. Baxter).

Method: Vehicle solution was prepared by mixing 1 volume of solution B,4 volumes of solution A, and 36 volumes of saline. The pH of theresulting vehicle solution is about 6. To prepare abis-N-acetyl-cysteine-trisulfide dosing solution at a concentration of30 mg/ml and about pH 6, the appropriate amount ofbis-N-acetyl-cystine-trisulfide was weighed into a clear glass vial andcooled to −20° C. Solutions B and A were mixed at a 1:2 volume ratio,vortexed, and cooled to 4° C. on ice. 1 mL of the ice-cold solution wasquickly added per 30 mg of bis-N-acetyl-cystine-trisulfide solid in thevial and vortexed immediately until the bis-N-acetyl-cystine-trisulfidesolid was completely dissolved. The pH was adjusted with 0.5N NaOH asneeded. The dosing solution was warmed to room temperature prior toparenteral administration. The dosing solution was stable for at least1.5 hours at room temperature.

Bis-N-acetyl-cystine-trisulfide andBis-N-methylsulfonamide-cystine-trisulfide exhibit unexpectedly highwater solubility compared to other polysulfides (Tables 1 and 2). Thisallows formulation in aqueous solution at high concentration, which isimportant for preparing parenteral dosage forms.

TABLE 1 Comparative Solubility Data Water 20% PEG400 17% HPBCD CompoundStructure (mg/mL) (mg/mL) (mg/mL)

0.037 0.11 >6

0.22 0.31 n.d.

0.10 0.24 n.d.

0.15 0.23 6.6

poor n.d. >20 (45% HPBCD)

poor n.d. 18

Not soluble in water, 90% PEG400, 45% HPBCD, 45% Captisol, 2% Tween 80,25% cremophor

>40 n.d. n.d.

>80 n.d. n.d. PEG, polyethylene glycol; HPBCD,hydroxypropyl-beta-cyclodextrin, n.d, no data.

TABLE 2 Comparative Vehicle Data Preferred Class Compound Name VehicleMW Allyl Polysulfide Di-allyl di-sulfide  

40% HPBCD/0.1% citric acid 146.27 Benzyl Polysulfide Mono-benzyl-mono-2-hydroxyethyl-tetrasulfide  

40% HPBCD/0.1% citric acid 264.45 Cysteine-trisulfideBis-N-acetyl-cysteine-trisulfide  

aqueous 356.44

Example 4 Increase of Biologically Available Sulfide in Blood and PlasmaThiosulfate by Intravenous Administration of Bis-N-acetylcystine-trisulfide

Pharmacokinetic experiments were conducted with groups of two to fouranimals consisting of 9-10 week old, male Sprague Dawley rats, 276-300grams (Charles River Laboratories, Wilmington, Mass.) with a jugularvein catheter and a femoral vein catheter. Animals were allowed toacclimate in a temperature and humidity controlled environment for 1-3days prior to the commencement of experimental procedures. Food andwater were provided ad libitum.

A baseline blood sample (˜0.4 mL) was collected from each rat throughthe jugular vein cannula into a heparin-coated syringe (1 mL volume)fitted with a 23G Luer stub adapter. After sampling, the blood volumewas replenished by slowly injecting 0.4 mL of a solution of 50 u/mLheparin in saline through the jugular vein cannula. A bolus dose ofbis-N-acetyl-cystine-trisulfide (e.g., 16 or 32 mg/kg IV in dosingsolution was injected through the femoral vein catheter. Bothbis-N-acetyl-cystine-trisulfide dosing solution and vehicle had a pH ofabout 6.0 (5.8 to 6.2). Blood (0.4 mL) was drawn at 0, 1, 5, 10, 30, 60& 120 minutes after dosing from the jugular vein cannula using aheparin-coated syringe (1 mL) with a 23G Luer stub adapter. Blood volumewas replenished as described above. Biologically available sulfide inblood and plasma thiosulfate were measured as described (Wintner et al.2010, Br. J. Pharmacology 160:941-957).

Intravenous administration of 16 or 32 mg/kgbis-N-acetyl-cystine-trisulfide leads to a dose-dependent elevation ofbiologically available sulfide in blood as well as an elevation ofplasma thiosulfate indicating that the compound delivers metabolizablesulfide to blood (see FIGS. 1 and 2). Elevation of blood sulfide can beextended by administration of repeated intravenous boli ofbis-N-acetyl-cystine-trisulfide as demonstrated in FIG. 6. Biologicallyavailable sulfide and plasma thiosulfate persists for 3 hours after twoconsecutive IV bolus injections of 20 mg/kgbis-N-acetyl-cystine-trisulfide at t=0 and t=15 min.

Example 5 No Elevation of Free Sulfide by Intravenous Administration ofBis-N-acetyl-cystine-trisulfide

Male CD rats (350-450 g, Charles River Laboratories) were anesthetizedby IV pentobarbital injection and the right jugular vein carefullyexposed and dissected away from the surrounding tissue. An amperometrichydrogen sulfide sensor (World Precision Instruments, Sarasota, Fla.,Cat. #ISO-H2S-2) was inserted into the jugular and advanced into thevena cava. Sensor current was monitored using a Powerlab 8/30(ADInstruments, Colorado Springs, Colo.). Once a stable baseline currentwas attained, the animals received either vehicle (pH matched saline) orbis-N-acetyl-cystine-trisulfide (20 mg/kg) administered over 1 min via aleft femoral vein catheter, and sensor current was monitored over 30min. Changes in current were calibrated against an IK-1001 standardcurve generated with the sensor in deoxygenated 50 mM HEPES, 150 mMNaCl, 1 mM DTPA at 37° C. and pH 7.4.

Intravenous injection of 20 mg/kg bis-N-acetyl-cystine-trisulfide doesnot lead to any measurable increase of free sulfide in blood, similar tothe injection of vehicle (FIG. 3). This result differs from the robustelevation of free sulfide observed after intravenous injection of a 1mg/kg sodium sulfide solution (Wintner et al. 2010, Br J Pharmacology160:941-957). Taken together with the data for mono-bromo-binamereactive sulfide, this experiment demonstrates thatbis-N-acetyl-cystine-trisulfide delivers biologically available sulfideto blood in a chemical form different from free sulfide.

The absence of free sulfide is reflected in the low toxicity of thesulfide-releasing compounds. The maximum tolerated dose in mice forintravenous injections of bis-N-acetyl-cystine-trisulfide was found tobe approximately 500 mg/kg, consistent with the absence of free sulfidein blood.

Example 6 Protection from Endotoxin-Induced Liver Damage byBis-N-acetyl-cysteine-trisulfide

Male C57BL/6 mice, (22 to 24 g, Charles River Laboratories) wereacclimated in a temperature and humidity controlled environment for 1-3days prior to commencement of the experimental procedures. All cageswere placed on water heating pads set at 30° C. Liver injury was causedby injecting mice intraperitoneally with 800 mg/kg d-Galactosamine(dGal), followed 30 min later by 100 μg/kg lipopolysaccharide (LPS)(FIG. 4) or with 1000 mg/kg dGal, followed 30 min later by 120 μg/kg LPS(FIG. 5). Bis-N-acetyl-cystine-trisulfide at 20 mg/kg (FIG. 4) or 40 and60 mg/kg (FIG. 5), or vehicle were injected intraperitoneally 30 minlater. Blood was collected by cardiac puncture under isofluraneanesthesia five hours after LPS injection. Serum was prepared andmarkers of liver injury, alanine aminotransferase (ALT) and aspartateaminotransferase (AST) measured. Bis-N-acetyl-cysteine trisulfideprotected from endotoxin-induced liver injury (FIGS. 4 and 5).

Example 7 Protection from Ischemia/Reperfusion-Induced Liver Damage byTrisulfide Test Compounds

C57BL/6 mice were anesthetized with a ketamine/xylazine combinationgiven via intraperitoneal injection. A midline laparotomy was performedto expose the viscera and the stomach and intestines were movedlaterally to allow for unimpeded visualization of the liver. Amicoaneurysm clip was applied to the hepatic artery and portal vein tocreate a partial liver ischemia encompassing the medial and left laterallobes (approximately 70% of the liver). In the last 5 minutes of a 45minute occlusion, vehicle or a test compound (5, 20, or 60 mg/kgbis-N-acetyl-cystine-trisulfide; or 2, 5, or mg/kgbis-N-methylsulfonyl-cystine-trisulfide) was injected into the inferiorvena cava. The aneurysm clips were removed at 45 minutes to restorehepatic blood flow which was confirmed visually. The abdominal incisionwas closed using wound clips and animals were recovered in a warmedchamber with 100% oxygen. Five hours after reperfusion, blood wascollected by cardiac puncture to measure serum alanine aminotransferase(ALT). Intravenous boli of 5 to 60 mg/kg bis-N-acetyl-cystine-trisulfideand of 2 to 20 mg/kg bis-N-methylsulfonyl-cystine-trisulfide protectedthe liver from ischemia/reperfusion injury (FIGS. 7 and 8).

Example 8 Generation of Biologically Available Sulfide in Blood byBis-N-acetyl-cystine-trisulfide and Inhibition of Formation byN-Aminoethyl-maleimide

The chemical nature of biologically available sulfide in blood wasprobed with the thiol-reactive reagent N-aminoethyl-maleimide (NAEM)which binds to free thiols and perthiols on proteins and blocks themfrom participating further in thiol-mediated chemical reactions.Biologically available sulfide was generated in blood by adding 1.3 mMbis-N-acetyl-cystine-trisulfide to 2 mL of whole rat blood. Biologicallyavailable sulfide was measured by derivatization with mono-bromo-bimaneand levels of bis-N-acetyl-cystine-trisulfide in blood were followed byLC/MS/MS. Bis-N-acetyl-cystine-trisulfide was consumed within 5 minuteswhile generating biologically available sulfide (FIG. 9). Pretreatmentof rat blood with 20 mM NAEM for 30 min prior to addition ofbis-N-acetyl-cystine-trisulfide reduced consumption ofbis-N-acetyl-cystine-trisulfide and almost completely blocked theproduction of biologically available sulfide. The concentration of NAEMwas chosen such that all of it was consumed within 1 min ofpreincubation with rat blood (FIG. 9), thus avoiding any potentialinterference with biologically-available sulfide production bybis-N-acetyl-cystine-trisulfide in blood. The experiment demonstratesthat biologically available sulfide created by exposure to sulfide donorreagents such as N-acetyl-cystine-trisulfide resides on thiols andperthiols in blood that can be blocked with NAEM.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A compound represented by the followingstructure:

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein m is 3or 4; Z is selected from H, CH₃, and CH₂CH₃; or, a salt, ester, hydrate,solvate, or stereoisomer thereof, with the proviso that when m is 3, Zis H, X and Y are C═O, R³ and R⁴ are CH₂, and R⁵ and R⁶ are OH, then R¹and R² are not C₁₋₁₀ alkyl.
 2. The compound of claim 1 wherein m is 3, Zis H, and R⁵ and R⁶ are OH.
 3. The compound of claim 2 wherein X and Yare SO₂, R¹ and R² are CH₃, and R³ and R⁴ are CH₂.
 4. The compound ofclaim 2 wherein X and Y are C═O and a) R¹ and R² are CH₃, and R³ and R⁴are C(CH₃)₂ or (CH₂)₂; b) R¹ and R² are CH₃, R³ is CH₂ and R⁴ isC(CH₃)₂; or c) R¹ is CH₃, R² is CH₂CH₃, and R³ and R⁴ are CH₂.
 5. Thecompound of claim 1 wherein m is 3, X and Y are C═O, Z is H and R¹ andR² are CH₃, R³ and R⁴ are CH₂, and, a) R⁵ and R⁶ are NHCH₂CO₂H; b) R⁵ isOH and R⁶ is NHCH₂CO₂H; or c) R⁵ is OH and R⁶ is NH(CH₂)₂ONO₂.
 6. Thecompound of claim 2 wherein X is C═O, Y is SO₂, R¹ and R² are CH₃, andR³ and R⁴ are CH₂.
 7. The compound of claim 1 wherein m is 4, Z is H,and R⁵ and R⁶ are OH.
 8. The compound of claim 1 wherein m is 3, X and Yare C═O, R¹ and R² are CH₃, R³ and R⁴ are CH₂, Z is CH₃, and, R⁵ and R⁶are OH.
 9. A compound represented by the structure:

wherein R¹⁰, R¹¹ and R¹² are each independently a side chain of anaturally-occurring or a non-naturally occurring amino acid, a branchedor straight chain C₁₋₁₀ alkyl, a straight or branched chain C₁₋₁₀ alkylsubstituted with —ONO₂, or, a straight or branched chain C₁₋₁₀ alkylsubstituted with —OH; wherein R¹³ and R¹⁴ are independently a branchedor straight chain C₁₋₁₀ alkanediyl; and, wherein X is independently C═Oor SO₂; or a compound represented by the structure:

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are each independently a side chain of anaturally occurring or a non-naturally occurring amino acid, a branchedor straight chain C₁₋₁₀ alkyl, a straight or branched chain C₁₋₁₀ alkylsubstituted with —ONO₂, or, a straight or branched chain C₁₋₁₀ alkylsubstituted with —OH; wherein R¹⁹ and R²⁰ are each independently abranched or straight chain C₁₋₁₀ alkanediyl; and, wherein each X isindependently C═O or SO₂; or, a salt, ester, hydrate, solvate orstereoisomer thereof.
 10. The compound of claim 9 wherein each X is C═O;R¹² and R¹⁸ are CH₃; and R¹⁰, R¹¹. R¹², R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are eachindependently a side chain of a naturally-occurring or a non-naturallyoccurring amino acid; or a salt, ester, free acid, hydrate, or solvateor stereoisomer thereof.
 11. A pharmaceutical composition comprising acompound selected from the group consisting of

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein m is 3or 4; Z is selected from H, CH₃, and CH₂CH₃;

wherein R¹⁰, R¹¹ and R¹² are each independently a side chain of anaturally-occurring or a non-naturally occurring amino acid, a branchedor straight chain C₁₋₁₀ alkyl, a straight or branched chain C₁₋₁₀ alkylsubstituted with —ONO₂, or, a straight or branched chain C₁₋₁₀ alkylsubstituted with —OH; wherein R¹³ and R¹⁴ are independently a branchedor straight chain C₁₋₁₀ alkanediyl; and, wherein X is independently C═Oor SO₂;

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are each independently a side chain of anaturally occurring or non-naturally occurring amino acid, a branched orstraight chain C₁₋₁₀ alkyl, a straight or branched chain C₁₋₁₀ alkylsubstituted with —ONO₂, or, a straight or branched chain C₁₋₁₀ alkylsubstituted with —OH; wherein R¹⁹ and R²⁰ are independently a branchedor straight chain C₁₋₁₀ alkanediyl; and, wherein X is independently C═Oor SO₂; and a salt, ester, hydrate, solvate or stereoisomer thereof; anda pharmaceutically acceptable vehicle, wherein the pharmaceuticalcomposition has a pH of 5.0 to 6.5.
 12. The pharmaceutical compositionof claim 11, which comprises a dosing concentration in the range of 2 to40 mg/ml.
 13. A method of making a linear symmetrical or asymmetricaltrisulfide compound represented by the structure:

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein Z isselected from H, CH₃, and CH₂CH₃; the method comprising: reacting acompound represented by the following structure

wherein R¹, X, R³ and R⁵ are as defined above, with an equal molaramount of 2,2′-thiodiisoindoline-1,3-dione in acetonitrile and water toproduce a ((1,3-dioxoisoindolin-2-yl)disulfanyl)-containingintermediate; isolating the intermediate; and reacting the intermediatewith a compound represented by the following structure

wherein R², X, R⁴ and R⁶ are as defined above, in an aqueous alcoholsolution, thereby forming the trisulfide compound.
 14. The method ofclaim 13, wherein the aqueous alcohol solution comprises 50%isopropanol.
 15. The method of claim 13 wherein the method comprisesreacting a compound represented by the structure:

with the 2,2′-thiodiisoindoline-1,3-dione, thereby forming thetrisulfide compound represented by the structure


16. The method of claim 13 wherein the compound which is reacted withthe 2,2′-thiodiisoindoline-1,3-dione is prepared by a method comprisingreacting a compound represented by the structure

with methanesulfonyl chloride to produce a reaction product representedby the structure

and treating the reaction product with triethylsilane andtrifluoroacetic acid to produce the compound.
 17. A method of making acyclic trisulfide compound represented by the structure:

wherein the method comprises preparing a dilute solution of a firstcompound represented by the structure

and 2,2′-thiodiisoindoline-1,3-dione in acetonitrile and water, andreacting the first compound and the 2,2′-thiodiisoindoline-1,3-dione toproduce the cyclic trisulfide compound.
 18. The method of claim 17wherein the concentration of the first compound in the dilute solutionis about 0.07 mM.
 19. A method of treating or preventing injury tobiological matter exposed to ischemic or hypoxic conditions comprisingadministering to a patient in need thereof a therapeutically effectivedose of a compound selected from the group consisting of

wherein R¹ and R² are independently a branched or straight chain C₁₋₁₀alkyl; a branched or straight chain C₁₋₁₀ alkyl substituted with —ONO₂;or, a branched or straight chain C₁₋₁₀ alkyl substituted with —OH;wherein R³ and R⁴ are independently a branched or straight chain C₁₋₁₀alkanediyl; wherein R⁵ and R⁶ are independently OH, an amino acidmoiety, or, a straight or branched chain C₁₋₁₀ amine substituted with—ONO₂; wherein X and Y are independently C═O or SO₂; and, wherein m is 3or 4; Z is selected from H, CH₃, and CH₂CH₃;

wherein R¹⁰, R¹¹ and R¹² are each independently a side chain of anaturally-occurring or a non-naturally occurring amino acid, a branchedor straight chain C₁₋₁₀ alkyl, a straight or branched chain C₁₋₁₀ alkylsubstituted with —ONO₂, or, a straight or branched chain C₁₋₁₀ alkylsubstituted with —OH; wherein R¹³ and R¹⁴ are independently a branchedor straight chain C₁₋₁₀ alkanediyl; and, wherein X is independently C═Oor SO₂;

wherein R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are each independently a side chain of anaturally occurring or a non-naturally occurring amino acid, a branchedor straight chain C₁₋₁₀ alkyl, a straight or branched chain C₁₋₁₀ alkylsubstituted with —ONO₂, or, a straight or branched chain C₁₋₁₀ alkylsubstituted with —OH; wherein R¹⁹ and R²⁰ are independently a branchedor straight chain C₁₋₁₀ alkanediyl; and, wherein X is independently C═Oor SO₂; and a salt, ester, hydrate, solvate or stereoisomer thereof. 20.The method of claim 19 wherein the hypoxic or ischemic conditions arerelated to reperfusion injury, tissue or organ transplantation, septicshock, cardiac arrest, exposure to cold or radiation, burns, wounds,amputations, stroke, hemorrhagic shock, surgical procedures, systemicinflammatory response syndrome, acute respiratory distress syndrome,kidney failure, liver failure or multiple organ failure.
 21. The methodof claim 19 wherein the compound is administered parenterally,intravenously, as a bolus or a repeated bolus.
 22. A((1,3-dioxoisoindolin-2-yl)disulfanyl)-containing compound representedby the following structure:

wherein R¹ is a branched or straight chain C₁₋₁₀ alkyl; a branched orstraight chain C₁₋₁₀ alkyl substituted with —ONO₂; or, a branched orstraight chain C₁₋₁₀ alkyl substituted with —OH; wherein R³ is abranched or straight chain C₁₋₁₀ alkanediyl; wherein R⁵ is OH, an aminoacid moiety, or, a straight or branched chain C₁₋₁₀ amine substitutedwith —ONO₂; wherein X is C═O or SO₂; and, wherein Z is selected from H,CH₃, and CH₂CH₃.